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Effects of Growth Factors on Proliferation and
Differentiation in Human Lens Epithelial Cells
in Early Subculture
Nobuhiro Ibaraki, Li-Ren Lin, and Venkat N. Reddy
Purpose. A successful method to subculture human lens epithelial (HLE) cells that retain
their intrinsic characteristics is of great importance. This study examines the effects of four
different growth factors on proliferation and differentiation in HLE cells in early subcultures.
Methods. Specimens of HLE cells were obtained from infants. First- or second-passage cells
were cultured in the presence of 10~2 to 102 ng/ml acidic and basic fibroblast growth factor
(aFGF, bFGF), epidermal growth factor (EGF), or insulin-like growth factor-I (IGF-I). Cell
proliferation was determined from cell number, and fiber differentiation was assessed from
the time of appearance, the number of lentoids formed, and the expression of y-crystallin.
Results. Cell proliferation was increased by EGF, bFGF, and IGF-I at concentrations greater
than 10"' ng/ml; the most effective concentration was 10 ng/ml. The effect of aFGF on
proliferation appeared only at a concentration of 102 ng/ml. EGF, bFGF, or IGF-I at 10 ng/
ml affected the time of appearance and the number of lentoids formed within 5 to 7 days.
In contrast, lentoids were observed after 42 days without the addition of growth factors.
Lentoid formation was accompanied by the expression of y-crystallin.
Conclusions. EGF, aFGF, bFGF, and IGF-I stimulated cell proliferation and fiber differentiation
in early subcultures. Invest Ophthalmol Vis Sci. 1995; 36:2304-2312.
A . successful method to subculture human lens epithelial (HLE) cells that retain their intrinsic characteristics is of great importance for the study of the normal
physiology of these cells and for investigations on the
etiology of cataract formation. There are numerous
studies in which several mammalian epithelial cells
have been cultured successfully for long periods of
time. 1 " 3 In contrast to the observations in animal lens
epithelial cells, HLE cells in tissue culture lose their
growth potential and their ability to differentiate into
fiber cells over a limited number of passages.4"6 Previous studies from this laboratory have shown that HLE
cells from infants can be grown successfully through
several passages, but their proliferative potency declines gradually.7 In addition, attempts in many laboraFrom the Eye Research Institute, Oakland University, Rochester, Michigan.
Supported in part by grants from the National Eye Institute, EY-00484 and EY05230 (Core Grant for Vision Research), and by Shojin Research Associate, Ltd.,
Los Angeles, California.
Submitted for publication February 22, 1995; revised fune 20, 1995; accepted fune
21, 1995.
Proprietary interest category: N.
Reprint requests: Nobuhiro Ibaraki, Eye Research Institute, Oakland University, 416
Dodge Hall, Rochester, MI 48309-4401.
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tories to culture lens epithelial cells from eye bank
lens or senile cataract have been less successful and
cannot be subcultured.8"11
In vitro fiber differentiation can be assessed from
the appearance of lentoids, which have the characteristics of lens fibers, gap junction formation, and the
expression of y-crystallin and membrane protein (MP26).12'13 Spontaneous in vitro fiber differentiation in
which lentoids arise from monolayer cultures has been
noted in most long-term cultures with HLE cells obtained from children and in early subcultures.1415
Also, fiber differentiation was observed in these shortterm cultures when the cells were grown on cell-substrate adhesion-free surfaces at high density.16
Fiber differentiation and crystallin expression in
lens epithelial cells of animals have been shown to be
affected by several kinds of growth factors,17"24 some of
which have been isolated from the retina, the vitreous
humor, and other ocular structures.25"30 Growth factor
is defined as a peptide that regulates cell proliferation
and differentiation similar to hormones.31 An epidermal
growth factor (EGF) receptor has been characterized in
rabbit lens epithelial cells using 125I-labeled EGF.32 Also,
Investigative Ophthalmology & Visual Science, October 1995, Vol. 36, No. 11
Copyright © Association for Research in Vision and Ophthalmology
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Growth Factor Effects on Cultured Human Lens Epithelial Cells
the effects of fibroblast growth factor (FGF) on rat lens
epithelial cells have been studied.20"22 This growth factor
has been found to regulate cell proliferation, migration,
and fiber differentiation in a concentration-dependent
manner.21 This capacity of FGF to modulate cell growth
and fiber differentiation decreases in cells obtained from
older rats.22 The effects of eye-derived growth factor1718
and retinal extracts19 on mammalian lens cells have been
attributed to FGF because anti-FGF antibody almost consistently prevents fiber differentiation induced by retinal
extracts.22 Other growth factors reported to affect cell
proliferation and differentiation on lens epithelial cells
in culture are insulin-like growth factor (IGF)2326 and
platelet derived growth factor.24
In contrast to the numerous studies with various
mammalian cells cited, there are no reports of the
effects of growth factors on HLE cells. In this study,
we describe the quantitative effect of four different
growth factors on proliferation in HLE cells in early
subcultures, and we report the enhancement of in
vitro differentiation of HLE cells as evidenced by lentoid formation, morphologic characteristics, and the
expression of y-crystallin, a marker protein for lens
cell differentiation.
MATERIALS AND METHODS
Culture of Human Lens Epithelial Cells
Specimens of HLE cells were obtained from 20 infants
3 to 9 months old who underwent surgery for retinopathy of prematurity. Informed consent was obtained
from parents or legal guardians, and Oakland University's institutional human experimentation committee
approval was granted. Tenets of the Declaration of
Helsinki were followed. The method for establishing
primary cultures has been described previously.7
Briefly, the pieces of capsule and epithelium obtained
during surgery were washed once with Ca 2+ -Mg 2+ free phosphate-buffered saline and collected with a
microsuction pipette. After microscopic examination,
small fragments of capsule (1 to 2 mm square) widi
epithelial cells attached were placed in a 60-mm culture dish (Falcon; Becton Dickinson, Oxnard, CA) as
explants.
Cells from primary culture or first-generation subculture were dissociated with trypsin-EDTA solution
(0.05% trypsin-0.02% ethylenediaminetetraacetic
acid solution; Gibco, Grand Island, NY), and inoculated at 70 ± 5 cells per mm2 of cell density in each
culture dish or protein-binding biopore membrane
(Millicell-HA; Millipore, Bedford, MA).
Cultures were incubated in a 5% CO2 atmosphere
at 36.5°C. Antibiotic-free Dulbecco's modified Eagle's
medium (Gibco) supplemented with 20% fetal bovine
serum (Gibco) was used as the standard medium. This
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medium has been shown to be optimal for maintenance of HLE cells in culture.7'33"34
Growth Factors
Human EGF (Sigma, St. Louis, MO), human FGFacidic (aFGF; Sigma), human FGF-basic (bFGF;
Sigma), and human IGF-I (IGF-I; Pepro Tech, Rocky
Hill, NY) were added to the standard medium. During
the culture period, the medium containing growth
factor was replaced three times per week.
Effect on Proliferation
To examine the effect of growth factors on cell proliferation, second-passage cells were selected because
proliferative potency of the cells had already decreased7 so that any stimulation with added growth
factors could be observed readily. Proliferation was
assessed by counting the cell number. Ten batches of
HLE cells from 10 infants were used; the cells from
the same batch were used for each experiment and
for each growth factor. The cells were seeded in three
wells for each culture period and each concentration
of growth factor using Multiwell tissue culture plate
(12-Well; Becton Dickinson, Lincoln Park, NJ). For
counting of the cells at four different time intervals
widi five different concentrations of each growth factor and controls, a total of 72 wells had to be cultured.
The experiments were repeated at least three times
for each growdi factor using different batches of cells.
To determine appropriate concentrations, growth factors were added to the medium at concentrations of
10~2, 1(T\ 1, 10, 102 ng/ml (1.7 X 10~12 to 1.7 X 10~8
M of EGF; 1.3 X 10"12 to 1.3 X 10"8 M of IGF-I; and
0.6 X 10"12 to 0.6 X 10~8 M of aFGF and bFGF). Cells
were dissociated with trypsin-EDTA solution and
counted using trypan blue and a hemocytometer at
various time periods from 1 day through 4 weeks after
incubation. The number of viable cells was counted
three times for each well.
To determine the quantitative proliferative potency, the maximum population doubling level
(PDLmax) was calculated.9'35 PDLmax is expressed as:
PDLmax = log10 (Nmax /No)/log10 2
where Nmax = maximum number of cells, and No =
initial number of cells.
Effect on Differentiation
For studies of fiber differentiation, we used first-passage cells because previous experiments indicated that
these cells had a greater capacity than cells in later
passages to undergo spontaneous in vitro differentiation36; the cells in second- or third-passage failed to
form lentoids at least during the 40- to 50-day culture
period. Differentiation was assessed from lentoid for-
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Investigative Ophthalmology & Visual Science, October 1995, Vol. 36, No. 11
mation, appearance of gap junctions in the lentoids,
and the expression of y-crystallin. Ten batches of HLE
cells derived from 10 infants were used; four batches
were used for the observations of lentoid formation,
and the other six were used for ultrastructural and
immunocytochemical studies. The cells on 60-mm
dishes were prepared for invert phase-contrast microscopy and transmission electron microscopy (TEM);
cells on 12-mm protein-binding membrane (MillicellHA) were used for immunogold labeling electron microscopy. The growth factors that affected cell proliferation were added to the standard medium. The concentration of EGF, bFGF, and IGF-I was 10 ng/ml (1.7
X 10"9, 0.6 X 10"9, and 1.3 X 10"9 M, respectively);
aFGF was 102 ng/ml (0.6 X 10~8 M).
Lentoid formation was observed under a phasecontrast microscope and quantified by counting the
total number of lentoids in the entire culture dish;
the cells from the same batch were used for each experiment, and at least two culture dishes were prepared for each growth factor in one experiment. This
experiment was repeated four times using four different batches of cells.
Transmission electron microscopy on monolayer
cultures was carried out in a fashion similar to the one
we previously described.16 Briefly, cells were cultured
for 14, 28, or 42 days and were rinsed several times
with serum-free Dulbecco's modified Eagle's medium
and fixed with 1% osmium tetroxide solution in 0.1
M cacodylate buffer (pH 7.4) for 3 minutes. After
fixation with 2.5% glutaraldehyde and 2% paraformaldehyde in cacodylate buffer for 3 hours, tissues were
refixed with 1% osmium tetroxide solution for 60
minutes. After each fixation, tissues were washed several times with cacodylate buffer. They were dehydrated through graded ethanols and embedded in
Poly/Bed 812 (Polysciences, Washington, PA). Sections approximately 60 nm thick were cut with a diamond knife, stained with 2% uranyl acetate and Reynolds' lead citrate solution, and examined with an ISILEM 2000 (International Scientific Instruments, Milpitus, CA) or a Phillips EM 410 (Phillips, Mahwah,
NJ) transmission electron microscope.
For immunogold labeling of y-crystallin, tissues
along with protein-binding membranes (MillicellHA) were fixed in 2% paraformaldehyde and 0.25%
glutaraldehyde in phosphate buffer (pH 7.4) for 1
hour on day 14 of cultivation. After dehydration
through graded ethanols, tissues were embedded in
L—R White resin (Polysciences), sectioned, and
mounted on nickel grids. Sections were blocked with
10% goat serum in 20 mM Tris-buffered saline with
0.1% bovine serum albumin (BSA-TBS, pH 7.4) for
1 hour, and then incubated with rabbit anti-human
y-crystallin antibody (1:100 dilution in BSA-TBS, pH
7.4) for 3 hours. This antibody has been shown to
react with y-crystallin but not with a- and /?-crystallin.716 After washing three times in BSA-TBS for a
total of 30 minutes and reblocking in goat serum in
BSA-TBS for 1 hour, the sections were treated in
goat anti-rabbit immunoglobulin containing 15 nm
colloidal gold (Amersham, Arlington Heights, IL;
1:100 dilution in BSA-TBS, pH 8.2) for 1 hour. The
grids were washed three times in BSA-TBS for a total
of 45 minutes, and they were dipped in distilled water
for 15 seconds and dried; they were then stained with
uranyl acetate and examined by TEM. Nonimmune
rabbit serum instead of y-crystallin antibody was used
as a negative control.
RESULTS
Cell Proliferation
During the course of this study, it became apparent
that the cells from different batches had varied growth
potential so that the results obtained from different
batches could not be pooled and compared. Therefore, a valid comparison could be made only between
controls and experimental data derived from the same
batch of cells.
The results of a typical experiment are shown in
Figure 1. All four growth factors studied affected cell
proliferation, and the effect of each growth factor on
cell number was concentration dependent. The increase in cell number was statistically significant after
5 days of cultivation and at a concentration greater
than 10"1 ng/ml of EGF, IGF-I, and bFGF; the most
effective concentration was 10 ng/ml (Student's Rest,
P < 0.001, w = 3 for each culture period and each
concentration). The effect of aFGF on proliferation
appeared only at a concentration of 102 ng/ml (Student's Hest, P < 0.005, n = 3 for each culture period
and each concentration). In addition to the increase
in cell number, the data in Figure 1 also show an
enhanced growth rate as indicated by the steeper
growth curves.
PDLmax is one of the quantitative mathematical
models to determine cell proliferative potency.9'35 Table 1 shows PDLmax of each concentration of the four
growth factors investigated in typical batches of cells.
PDLmax was significantly higher with IGF-I and EGF
compared to their controls at a concentration of 10~2
ng/ml, whereas bFGF and aFGF required concentrations greater than 10"' and 102 ng/ml, respectively
(Student's West, P < 0.001 for EGF, IGF-I, and bFGF,
P < 0.005 for aFGF, n = 6 for each culture period
and each concentration).
Similar results to those shown in Figure 1 and
Table 1 were obtained in other experiments in which
cells from different batches were used (data not
shown).
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Growth Factor Effects on Cultured Human Lens Epithelial Cells
FIGURE 1. The effect of growth
factors on cell proliferation,
(a) EGF, (b) IGF-I, (c) aFGF,
(d) bFGF. Growth factors were
added to the standard medium
( 0 ) at concentrations of 102
(•), 10 (O), 1 ( • ) , 10"' (A),
10~2 (T) ng/ml. Cell proliferation was stimulated with more
than 10"1 ng/ml of b-FGF,
EGF, IGF-I (P < 0.001), and
102 ng/ml of a-FGF (P <
0.005). n = 3 for each culture
period and each concentration. Student's t-test was used.
Standard deviations are omitted to simplify these graphs.
2307
7
14
21
26
Culture Period (days)
Culture Period (days)
7
14
21
Culture Period (days)
Culture Period (days)
ferent culture periods. In a typical experiment, as
shown in Figure 3, three culture dishes were prepared
for each growth factor. Lentoids appeared from day
5 with EGF, day 7 with bFGF and IGF-I, and day 14
with aFGF. The number of lentoids increased gradually and reached 451 ± 21 with EGF, 250 ± 15 with
bFGF, 70 ± 7 with IGF-I, and 22 ± 1 with aFGF on
day 42. In contrast, only 4 ± 0 lentoids were observed
after 42 days without the addition of growth factors.
The number of lentoids formed in cells from different
batches varied between each culture period and with
each growth factor, but the time course showed similar
results for all batches of cells (data not shown).
The ultras true ture of lentoids enhanced with EGF
is shown in Figure 4. Lentoids consisted of multilayers
of enlarged cells that had few cytoplasmic organelles
on day 14. On day 28, the cytoplasm was very finely
granular and uniform in density, with concomitant
Cell Differentiation
In addition to the effect on cell proliferation, the
growth factors had an influence on the time of appearance and the number of lentoids formed. Figure 2
shows phase-contrast micrographs of first-passage cells
with 10 ng/ml of EGF, bFGF, and IGF-I and 102 ng/
ml of aFGF. With EGF, cell size became smaller than
control cells, cell aggregations appeared on day 3 after
cultivation, and lentoids formed on day 5. The cells
surrounding the lentoid became larger than the cells
in the early culture period. With bFGF or IGF-I, cell
aggregation first appeared on day 5, and lentoid formations were observed on day 7. With aFGF, cell aggregation appeared on day 7, and no lentoids were
seen until day 14. The time course and the number
of lentoids formed in the presence of growth factors
were determined by counting the total number of lentoids present in the entire 60-mm culture dish at dif-
TABLE l. Population Doubling Level Maximum (PDLmax) of Human Lens Epithelial Cells
With Growth Factors
Concentration(ng/ml)
Growth Factors
EGF
IGF-I
aFGF
bFGF
102
2.74 ±
1.92 ±
2.48 ±
2.68 ±
0.00*
0.04*
O.Olf
0.06f
1
10
3.42 ± 0.05*
2.73 ± 0.15*
1.88 ± 0.09
3.55 ± 0.29*
2.92
2.58
2.14
3.32
± 0.02*
± 0.07*
± 0.02
± 0.05*
10 "'
2.19 ±
2.09 ±
1.75 ±
3.02 ±
0.11*
0.02*
0.22
0.18*
10~2
0 (control)
2.08 ± 0.00*
2.00 ± 0.12*
1.72 ± 0.02
2.04 ± 0.12
0.75 ± 0.30
0.75 ± 0.30
1.91 ± 0.09
1.91 ± 0.09
EGF = epidermal growth factor; IGF = insulin-like growth factor; aFGF = acidic fibroblast growth factor; bFGF = basic fibroblast
growth factor. The method to calculate PDLmax is given in the text. Briefly, maximum number of cells for each concentration and
each growth factor was used for calculating PDLmax. Two different batches of cells, one for EGF and IGF-I and the other for aFGF and
bFGF, were used in this experiment.
Values are means ± SD. n = 6. Statistical analysis performed using Student's t-test.
*P< 0.001; f P < 0.005.
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Investigative Ophthalmology & Visual Science, October 1995, Vol. 36, No. 11
y-crystallin. Even in the lentoid formed on day 14, an
early stage of fiber differentiation, many gold particles
were observed in the cytoplasm. In contrast, no gold
particles were observed in the negative control, and
there was little reaction against y-crystallin in monolayers of HLE cells (nonlentoid).
DISCUSSION
The major purpose of this investigation was to examine the possible effects of a number of growth factors
in HLE cells to establish a more successful culture
system than currently available. All the growth factors
selected for this study are known to stimulate proliferation of cells of ectodermal origin.31
The results of this study clearly demonstrate that
a number of growth factors enhance the proliferative
potency of HLE cells in culture and stimulate fiber
differentiation, as evidenced by the time course and
the number of lentoids formed. These findings are
similar to the observations made in a number of cells
derived from lenses of various animals. 17~2* Our observations in HLE cells have been made on early subcultures that had a low but definite proliferative potency.
Because of die limited number of cells that can be
derived from each specimen and the varied proliferative potency of HLE cells, the results of cell proliferation enhanced by growth factors could only be statistically compared with the data obtained from the same
batch of cells. Based on population doubling criteria
(PDLmax), EGF was most effective in promoting cell
proliferation, and the effect was dose dependent. The
80 n
FIGURE 2. The effect of growth factors on lentoid formation
as seen by phase-contrast microscopy, (a) The standard medium, (b) EGF, (c) IGF-I, (d) aFGF, and (e) bFGF. Arrows
indicate lentoids, and arrowhead shows cell aggregation.
loss of cytoplasmic organelles and the appearance of
interdigitations or interlocking processes. In some
areas, typical gap junctions were observed under
higher magnification. Similar observations were made
with bFGF and IGF-I (data not shown). In contrast,
there were only normal epithelial cells, even on day
28, and interdigitations, gap junctions, and homogeneous cytoplasms appeared after 42 days of cultivation
in the standard medium.
Figure 5 is a transmission electron microscopic
view of lentoid sections labeled with immunogold for
5
7
14
21
Cutlure Period (days)
FIGURE 3. The effect of growdi factors on the time course
and number of lentoids formed. Lentoids appeared on day
5 with 10 ng/ml of EGF (O), day 7 with 10 ng/ml of IGFI (A) and bFGF (•), and day 14 with 102 ng/ml of aFGF
(•). The lentoid number increased gradually, and the mean
number reached 451 with EGF, 250 with bFGF, 70 with IGFI, and 22 with aFGF on day 42. In contrast, four lentoids
were observed after 42 days in the control culture ( 0 ) .
Standard deviations are given in the text.
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Growth Factor Effects on Cultured Human Lens Epithelial Cells
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FIGURE 4.
The effect of growth factors on cell differentiation as seen by transmission electron
microscopy. Lentoid formation stimulated with 10 ng/ml of EGF consisted of multilayered,
elongated cells that had few cytoplasmic organelles on day 14 (b). On day 28, interdigitations
(arrowheads) and fine granular cytoplasms with few organelles were observed (c); at higher
magnification (d); gap junctions (arrows) were clearly evident. In control, there were only
normal epithelial cells even on day 28 (a).
effect of the other growth factors, bFGF and IGF-I,
was also concentration dependent. These findings are
similar to those with bFGF on rat lens epithelial cells.21
We used 20% fetal bovine serum in the standard
medium because HLE cells require at least 5% of fetal
bovine or calf serum to be alive or to show any growth;
the optimum serum concentration is 15% to 20%.33"s'1
Even at the optimum concentration of serum, the proliferative capacity of HLE cells is very low compared
with many animal lens epithelial cells.1"11 However,
the addition of growth factors used in this study significandy enhanced cell proliferation. To the extent
that serum is thought to be associated with some
growth factors of undetermined nature and unknown
quantity, there is a possibility that our results may represent the combined effects of the added growth factors and those that might be present in serum or other
unknown factors in serum. In many tissues, growth
factors have been shown to have multiple effects; they
have been found to be mitogenic and inhibitory as
well as to promote cell survival.31 At the highest con-
centration used—102 ng/ml—these growdi factors
had little effect on PDLmax and growth curves compared with the effects noted with 1 ng/ml of EGF,
and 10"' ng/ml of bFGF or IGF-I. It has been reported
that cell proliferation in rat lens epithelial cell culture
is optimized by lower concentrations of bFGF than
fiber differentiation.121 In our study, only one concentration of each growth factor, which was determined
as the optimum for cell proliferation, was used for
fiber differentiation. It is possible that the number of
lentoids formed may increase with higher concentrations of growth factors if die human lens system behaves similarly to the rat lens epithelium, but this remains to be established.
In vitro fiber differentiation of HLE cells in this
study was assessed from morphologic characteristics
of lentoids as well as y-crystallin expression. In contrast to the spontaneous fiber differentiation in controls, without the addition of growth factors, in which
lentoids are few and appeared after long periods of
culture, EGF, aFGF, bFGF, and IGF-I affected both
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Investigative Ophthalmology & Visual Science, October 1995, Vol. 36, No. 11
FIGURE 5. Immunogold labeling of y-crystallin. On day 14, many gold particles were observed
in lentoids formed in the presence of 10 ng/ml of bFGF (a). In contrast, no gold particles
were found in lentoid, in which nonimmune serum instead of y-crystallin antibody was used
(b). There was little reaction with y-crystallin antibody in the monolayers of the cultured
cells with the standard medium (c). N = cell nucleus; asterisks = cellular membrane.
the time of appearance and the number of lentoids
formed. The time course of the morphologic changes
of these lentoids included the decrease of cytoplasmic
organelles, the formation of interdigitations or interlocking of cellular membranes, the appearance of a
very finely granular and uniform density structure with
concomitant loss of cytoplasmic organelles, and the
presence of abundant gap junctions. These findings
are consistent with the differentiation of lens epithelial cells into fiber-like structures,16 and the results
clearly indicate that, in addition to their stimulatory
effect on cell proliferation, growth factors also enhance lens cell differentiation. Although the presence
of gap junctions in lentoids as seen by TEM is consistent with fiber differentiation, the identity of these
structures with gap junctions in situ remains to be
established by other criteria. This ability of growth
factors to stimulate or accelerate lentoid formation
may prove useful in studying the HLE cell differentiation in vitro. Although previous studies17"23 on animal
lens cells have shown that growth factors enhance cell
differentiation, no detailed morphologic characteristics of the fibers formed were presented that could be
compared with in situ structures.
In the current study, y-crystallin, a marker protein
for fiber differentiation,1213 was detected in lentoids
on day 14 of culture when the morphologic characteristics showed only loss of cytoplasmic organelles.
These results suggest that y-crystallin expression is induced at an early stage of fiber differentiation, i.e.,
before the appearance of interdigitations or gap junctions.
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Growth Factor Effects on Cultured Human Lens Epithelial Cells
Growth factors have been detected in human ocular media. 27 " 30 Furthermore, human ocular media
consisted of a mixture of growth factors. At least in
rat lens epithelial cell cultures, IGF-I was shown
to enhance bFGF-induced fiber differentiation.37
Whether multiple growth factors influence cell differentiation in HLE cells is unknown and remains a subject for further study.
KeyWords
cell proliferation, epidermal growth factor, fiber differentiation, fibroblast growth factor, y-crystallin expression, gap
junction, human lens epithelial cells, insulin-like growth factor, lentoid
Acknowledgments
The authors thank Dr. Michael Trese of the Eye Research
Institute and William Beaumont Hospital (Royal Oak, Michigan) for his cooperation in obtaining specimens of human
lens epithelium, and Dr. J. Samuel Zigler, Jr. of the National
Eye Institute for the antisera for 7-crystallin.
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